Unicellular and multicellular developmental variations in algal zygotes produce sporophytes.

The emergence of sporophytes, that is, diploid multicellular bodies in plants, facilitated plant diversification and the evolution of complexity. Although sporophytes may have evolved in an ancestral alga exhibiting a haplontic life cycle with a unicellular diploid and multicellular haploid (gametophyte) phase, the mechanism by which this novelty originated remains largely unknown. Ulotrichalean marine green algae (Ulvophyceae) are one of the few extant groups with haplontic-like life cycles. In this study, we show that zygotes of the ulotrichalean alga Monostroma angicava, which usually develop into unicellular cysts, exhibit a developmental variation producing multicellular reproductive sporophytes. Multicellular development likely occurred stochastically in individual zygotes, but its ratio responded plastically to growth conditions. Sporophytes showed identical morphological development to gametophytes, which should reflect the expression of the same genetic programme directing multicellular development. Considering that sporophytes were evolutionarily derived in Ulotrichales, this implies that sporophytes emerged by co-opting the gametophyte developmental programme to the diploid phase. This study suggests a possible mechanism of sporophyte formation in haplontic life cycles, contributing to the understanding of the evolutionary transition from unicellular to multicellular diploid body plans in green plants.

Mitochondrial uniparental inheritance achieved after fertilization challenges the nuclear-cytoplasmic conflict hypothesis for anisogamy evolution.

In eukaryotes, a fundamental phenomenon underlying sexual selection is the evolution of gamete size dimorphism between the sexes (anisogamy) from an ancestral gametic system with gametes of the same size in both mating types (isogamy). The nuclear-cytoplasmic conflict hypothesis has been one of the major theoretical hypotheses for the evolution of anisogamy. It proposes that anisogamy evolved as an adaptation for preventing nuclear-cytoplasmic conflict by minimizing male gamete size to inherit organelles uniparentally. In ulvophycean green algae, biparental inheritance of organelles is observed in isogamous species, as the hypothesis assumes. So we tested the hypothesis by examining whether cytoplasmic inheritance is biparental in Monostroma angicava, a slightly anisogamous ulvophycean that produces large male gametes. We tracked the fates of mitochondria in intraspecific crosses with PCR-RFLP markers. We confirmed that mitochondria are maternally inherited. However, paternal mitochondria enter the zygote, where their DNA can be detected for over 14 days. This indicates that uniparental inheritance is enforced by eliminating paternal mitochondrial DNA in the zygote, rather than by decreasing male gamete size to the minimum. Thus, uniparental cytoplasmic inheritance is achieved by an entirely different mechanism, and is unlikely to drive the evolution of anisogamy in ulvophyceans.

Evolution of Anisogamy in Organisms with Parthenogenetic Gametes.

AbstractThe two sexes are defined by the sizes of the gametes they produce, anisogamy being the state with two differing gamete sizes (hence, females and males). The origin of this divergence has received much research interest, both theoretically and empirically. The gamete dynamics (GD) theory is a widely accepted theoretical explanation for anisogamy, and green algae have been an important empirical testing ground for the theory. However, some green and brown algae produce parthenogenetic gametes (gametes that can develop without fusing with another gamete), in contrast to an assumption in GD theory that unfused gametes do not develop. Here, we construct a GD model accounting for parthenogenetic gametes. We find that under conditions of panmixia and highly efficient fertilization (i.e., conditions of classical GD models from 1972 onward), the results remain largely unaltered by parthenogametes. However, under gamete-limited conditions anisogamy evolves less easily in the new model, and a novel result emerges: whereas previous models typically predict the evolution of either anisogamy or small isogamy, the current model shows that large isogamy can evolve when parthenogenetic gametes evolve under conditions of inefficient fertilization. Our analyses uncover unexplored complications relating to sex ratios under this relatively uncharted gametic system. We discuss limitations these complications impose on our models and suggest avenues for future research. We compare model results to algae with parthenogenetic gametes in nature.

Identification of genomic differences between the sexes and sex-specific molecular markers in Monostroma angicava (Ulvophyceae).

There is little information available regarding genomic differences between sexes in ulvophycean green algae. The detection of these differences will enable the development of sex-discriminating molecular markers, which are useful for algae showing little apparent difference between sexes. In this study, we identified male- and female-specific DNA sequences in the ulvophycean marine green alga Monostroma angicava, which has a dioicous heteromorphic haplo-diplontic life cycle, via next-generation sequencing. Fluorescence in situ hybridization (FISH) showed that signals for the sex-specific sequences exist only in the nuclei of the corresponding sex, confirming the specificity of the sequences. Sex-specific molecular markers that targeted these sequences successfully distinguished the sex of gametophytes even in geographically distant populations, indicating that the sex-specific sequences are universal. These results consistently suggest that male and female gametophytes of M. angicava are genetically different, implying that sex may be determined genetically in this alga.

Nuclear behavior and roles indicate that Codiolum phase is a sporophyte in Monostroma angicava (Ulotrichales, Ulvophyceae).

The life-cycle system of Ulotrichales, a major order of Ulvophyceae, remains controversial because it is unclear whether the Codiolum phase, a characteristic unicellular diploid generation in ulotrichalean algae, is a zygote or a sporophyte. This controversy inhibits the understanding of the diversified life cycles in Ulvophyceae. To distinguish between zygotes and sporophytes, we have to examine not only whether diploid generations function as sporophytes, but also whether mitosis occurs before meiosis in diploid generations. However, the nuclear behavior in the Codiolum phases is largely unknown, probably because no suitable methods are available. Using fluorescent microscopy with ethidium bromide and transmission electron microscopy of cell-wall-dissected specimens, we report the nuclear behavior in the Codiolum phases of an ulotrichalean alga with a representative life cycle, Monostroma angicava. Each vegetative Codiolum phase had a single polyploid nucleus due to endoreduplication, a type of mitosis without nuclear division. During zoosporogenesis, the nucleus had a structure that would be a meiosis-specific complex. We quantitatively showed that Codiolum phases grew extremely large and produced numerous zoospores. Our results suggest that an event comparable to mitosis occurs before meiosis in the Codiolum phase of M. angicava. This nuclear behavior and the functions (growth and zoospore production abilities) correspond to those of sporophytes. Therefore, the life-cycle system of M. angicava is a heteromorphic haplo-diplontic cycle. This system appears to be widely adopted among other ulotrichalean algae.

Onset of a limit cycle and universal three-body parameter in Efimov physics.

The Efimov effect is the only experimentally realized universal phenomenon that exhibits the renormalization-group limit cycle with the three-body parameter parametrizing a family of universality classes. Recent experiments in ultracold atoms have unexpectedly revealed that the three-body parameter itself is universal when measured in units of an effective range. By performing an exact functional renormalization-group analysis with various finite-range interaction potentials, we demonstrate that the onset of the renormalization-group flow into the limit cycle is universal, regardless of short-range details, which connects the missing link between the two universalities of the Efimov physics. A close connection between the topological property of the limit cycle and few-body physics is also suggested.

A comparative test of the gamete dynamics theory for the evolution of anisogamy in Bryopsidales green algae.

Gamete dynamics theory proposes that anisogamy arises by disruptive selection for gamete numbers versus gamete size and predicts that female/male gamete size (anisogamy ratio) increases with adult size and complexity. Evidence has been that in volvocine green algae, the anisogamy ratio correlates positively with haploid colony size. However, green algae show notable exceptions. We focus on Bryopsidales green algae. While some taxa have a diplontic life cycle in which a diploid adult (=fully grown) stage arises directly from the zygote, many taxa have a haplodiplontic life cycle in which haploid adults develop indirectly: the zygote first develops into a diploid adult (sporophyte) which later undergoes meiosis and releases zoospores, each growing into a haploid adult gametophyte. Our comparative analyses suggest that, as theory predicts: (i) male gametes are minimized, (ii) female gamete sizes vary, probably optimized by number versus survival as zygotes, and (iii) the anisogamy ratio correlates positively with diploid (but not haploid) stage complexity. However, there was no correlation between the anisogamy ratio and diploid adult stage size. Increased environmental severity (water depth) appears to drive increased diploid adult stage complexity and anisogamy ratio: gamete dynamics theory correctly predicts that anisogamy evolves with the (diploid) stage directly provisioned by the zygote.

Characteristics of mitosis in the gametophyte cells of the marine green alga Monostroma angicava.

BACKGROUND: Some marine algae exhibit several characteristics of mitosis (e.g., the timing of mitosis such as diurnal periodicity) that are unique from those of land plants. Not only the timing but also other characteristics of mitosis, including the process itself and the number of chromosomes involved, are largely unknown in ulvophycean marine green algae. Effective mitotic inhibitors are useful for observing mitosis and identifying the number of chromosomes. However, few such inhibitors are available for ulvophycean algae. Here, we examined the timing and process of mitosis and the number of chromosomes with several mitotic inhibitors in the haploid gametophyte cells of the Ulvophyceae alga Monostroma angicava. RESULTS: Mitosis did not occur during the light period but began immediately after the onset of the dark period. The typical process of mitosis was observed. The mitotic inhibitors colchicine and 8-hydroxyquinoline, which generally arrest mitosis in land plants, were ineffective in M. angicava. We found that three other mitotic inhibitors, amiprophos methyl, griseofulvin and oryzalin, are effective to arrest mitosis. With three-dimensional fluorescence microscopy, we demonstrated that there were nine chromosomes in each cell. CONCLUSIONS: In the gametophyte cells of M. angicava, mitosis occurs diurnally. It is triggered by the onset of the dark period. We identified the number of chromosomes as N = 9. Our study shows effective inhibitors to observe mitosis in ulvophycean algae.

Within-clutch variability in gamete size arises from the size variation in gametangia in the marine green alga Monostroma angicava.

KEY MESSAGE: Within-clutch gamete size variability in Monostroma angicava. In many organisms, it is unclear how the size variation in gametes is generated in each clutch (i.e., total gametes produced by a gametophyte for a single spawning) or how gamete size is adjusted. Within-clutch variation in gamete size has been explained as a result of either physiological/developmental constraints or bet hedging during gametogenesis. These two explanations have been assumed to be mutually exclusive, and related observations are conflicting. The slightly anisogamous dioecious green alga Monostroma angicava employs a simple mechanism to produce gametes of each sex: each vegetative cell becomes a single gametangium cell, which synchronously divides to form equally sized gametes. The number of such cell divisions has several variations, which might vary gamete size. We measured the volume of gametangia in each clutch, counted the number of cell divisions in each gametangium and estimated the size of the gametes. We found that larger gametangia divided more times than smaller gametangia in both sexes, although male gametangia were smaller than female gametangia when they underwent the same number of cell divisions. Therefore, the variation in the number of cell divisions during gametogenesis serves to adjust gamete size in each sex rather than to vary it. Within-clutch gamete size variability originates in within-clutch variation in gametangium size: any factors that increase the variation in the size of gametangia can increase the within-clutch variation in gamete size.

Evidence for equal size cell divisions during gametogenesis in a marine green alga Monostroma angicava.

In cell divisions, relative size of daughter cells should play fundamental roles in gametogenesis and embryogenesis. Differences in gamete size between the two mating types underlie sexual selection. Size of daughter cells is a key factor to regulate cell divisions during cleavage. In cleavage, the form of cell divisions (equal/unequal in size) determines the developmental fate of each blastomere. However, strict validation of the form of cell divisions is rarely demonstrated. We cannot distinguish between equal and unequal cell divisions by analysing only the mean size of daughter cells, because their means can be the same. In contrast, the dispersion of daughter cell size depends on the forms of cell divisions. Based on this, we show that gametogenesis in the marine green alga, Monostroma angicava, exhibits equal size cell divisions. The variance and the mean of gamete size (volume) of each mating type measured agree closely with the prediction from synchronized equal size cell divisions. Gamete size actually takes only discrete values here. This is a key theoretical assumption made to explain the diversified evolution of isogamy and anisogamy in marine green algae. Our results suggest that germ cells adopt equal size cell divisions during gametogenesis.